SUMMARY Squamous cell carcinoma (SCC) accounts for over 90% of head and neck (HN) cancer. HNSCCs in heavy smokers respond poorly to therapies and have the highest rate of relapse/recurrence among all HNSCC patients. Inhibitors of programmed death-1 (PD-1) are FDA approved to treat relapsed/recurrent HNSCCs, but are only effective in ~25% of HNSCCs, indicating additional immune suppressive/evasion mechanisms. We reported that transforming growth factor-?1 (TGF?1), an immune suppressor, is elevated in >60% of tobacco- associated HNSCCs. Unique to HNSCC, TGF?1 causes excessive inflammation with the majority of tumor infiltrating leukocytes being myeloid cells. Developing new therapeutic interventions that effectively target these tumor microenvironment (TME) characteristics is hindered by a dearth of HNSCC models with metastatic potential in an immune competent background. This application employs new therapeutic agents to target both PD-L1 and TGF? in HNSCC and metastasis, and analyze the underlying mechanisms. We have created a mouse model in which Smad4, a tumor suppressor frequently lost in tobacco-associated HNSCCs, is deleted (Smad4-/-) in head and neck epithelia. Smad4 loss causes SCC and metastasis, and compensatory TGF?1 overproduction. Preliminary data revealed that Smad4-/- SCCs also overexpress PD-L1 and short-term TGF? inhibition sensitized SCCs to anti-PD-L1. Further, in mice with SCC eradicated, re-transplanting the same SCC cell line failed to initiate new tumors, suggesting a memory T cell-dependent anti-tumor response. TGF? inhibition also reduced SCC lung metastases in immune compromised mice. Taken together, we hypothesize that attenuating a TGF?-induced immune suppressive and inflammatory TME in Smad4 mutant HNSCCs makes immunotherapy more effective, thus dual TGF??PD-L1 inhibition eradicates these HNSCCs via T-cell-dependent and -independent mechanisms. Aim 1 will perform experimental therapeutics using novel TGF?/PD-L1 inhibitor drugs on genetic mouse models and transplanted human HNSCCs to determine if Smad4 loss and TGF?1 overexpression predict therapeutic response to TGF?/PD-L1 dual inhibition in HNSCCs in immune competent and compromised conditions. Aim 2 will assess T cell- dependent mechanisms of TGF? inhibition on sensitizing or synergizing with anti-PD-L1-mediated SCC eradication, utilizing tumors generated in Aim 1 and patient HNSCC specimens to examine if Smad4 loss and TGF?/PD-L1 levels correlate with immune suppressive T cell profiles. Aim 3 will use tumors generated in Aim 1 to assess if myeloid cell-dependent targeting effects of TGF?/PD-L1 inhibition contribute to therapeutic efficacy in HNSCC and metastasis, and patient HNSCC specimens to examine if Smad4 loss and TGF?/PD-L1 levels correlate with increased myeloid cells and associated molecular markers. These studies will lead to a novel therapeutic strategy for HNSCC patients with high rates of recurrence and metastasis. Additionally, the mechanistic studies will offer novel insights into future biomarker-driven selection for future clinical trials of TGF?/PD-L1 dual inhibition in HNSCC patients.